Anti etv1

Total protein extraction from cells using lysis buffer (absin, Shanghai, China). Cellular proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane by electroblotting. Following blocking with a 5% nonfat dry milk solution for 2 hours, the membrane was incubated overnight at 4°C with the appropriate primary antibody (1 : 1000 dilution). Finally, the membrane was incubated with the corresponding secondary antibody (1 : 5000 dilution) for 2 h at room temperature. The anti-c-KIT antibody was obtained from Invitrogen. Anti-p-ERK (Y204), anti-ERK, and anti-GAPDH were obtained from Cell Signaling Technology. Anti-ETV1 was obtained from Abcam. Secondary antibodies were purchased from Absin.

All DNA constructs were made in the corresponding author’s laboratory and verified by DNA sequencing and/or restriction enzyme analysis. Chemicals were purchased at molecular biology or analytical grade purity from established vendors (e.g., Sigma-Aldrich, VWR). Enzymes were obtained from Promega or New England Biolabs. The following antibodies were used for western blotting or immunoprecipitation: anti-Flag M2 (Sigma-Aldrich F1804), anti-Myc 9E10 (Sigma-Aldrich M4439), anti-cyclin D1 DCS6 (Cell Signaling #2926), anti-SMAD4 B-8 (Santa Cruz sc-7966) and anti-actin (GenScript A00730) mouse monoclonal antibodies; anti-ETV1 (Abcam ab81086 or our previously described #959 antibody^{9 (link)}), anti-p21 H-164 (Santa Cruz sc-756), anti-PMEPA1 2A12 (Abnova H00056937-M01) and anti-SMAD3 (Zymed 51–1500) rabbit polyclonal antibodies; and anti-p-SMAD3-Ser423/425 C25A9 (Cell Signaling #9520) rabbit monoclonal antibodies.